The foreign body response (FBR) represents a major challenge in the application and clinical success of current and future biomaterial-based treatments of musculoskeletal injuries and diseases. The FBR is orchestrated by macrophages and occurs ubiquitously to all implanted non-biological materials. Although the mechanisms driving the FBR remain to be elucidated, it is generally understood the FBR is initiated by inflammatory cells that recognize the material as foreign through surface-adsorbed proteins, which can unfold, exposing epitopes known as damage-associated molecular patterns (DAMPs). These DAMPs are directly influenced by the highly dynamic and heterogeneous behavior of proteins in near-surface environments, including transient unfolding and refolding, rapid exchange of folded and unfolded protein molecules between the surface and bulk solution, and intermittent diffusion on the surface. While such interfacial processes are likely involved in the FBR, their roles have been all but ignored due to the lack of experimental techniques to directly observe these processes. The overarching aim of this research is to investigate the extent to which interfacial protein dynamics influences the presence of DAMPs using novel single-molecule (SM) biophysical methods, which are uniquely sensitive to interfacial protein dynamics. Such methods will be combined with poly(ethylene glycol)/poly(sulfobetaine) copolymer brushes that are tuned to control interfacial dynamic behavior of proteins, in vitro macrophage activation assays, and in vivo mouse studies to elucidate the role of surface-induce protein unfolding in the FBR. We will test the hypothesis that the presentation of unfolded proteins (i.e., DAMPs) as a result of the complex and heterogeneous behavior of proteins in near-surface environments triggers macrophage activation via toll like receptor (TLR) signaling and contributes to the FBR. In particular, toll-like receptors (TLRs) are a class of membrane proteins that are involved in innate immune signaling; and specifically, TLR2 and 4 have been shown to recognize host proteins acting as DAMPs. Thus, in Aim 1, we will confirm the role of TLR2/4 signaling in the activation of macrophages to known DAMPs (Aim 1.1) and identify the dynamic behaviors that lead to the presentation of unfolded proteins and in turn to macrophage activation (Aim 1.2). In Aim 2, the amount of transient unfolded protein will be further correlated to the FBR in vivo via TLR2/4 signaling using knockout mouse models. Combined, these aims will 1) identify that transient unfolded proteins contribute to macrophage activation and the FBR via TLR2/4 signaling and 2) elucidate the mechanisms by which near surface environments influence transient protein unfolding. These new insights will provide the foundation for new research aimed at designing novel chemistries that control complex protein dynamics and thus we may, for the first time, be able to prevent protein unfolding at surfaces and potentially eliminate the FBR.